RU2410425C1 - Culture medium for growing mycobacterium tuberculosis - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in diagnosis of tuberculosis. The culture medium contains potassium phosphate, magnesium sulphate, magnesium citrate, L-asparagine, glycerin, potato starch, distilled water, aqueous extract from powder of Siberian deer, aqueous 2% solution of malachite green and egg mixture.

EFFECT: invention shortens duration of isolating mycobacteria.

4 tbl, 2 ex

Description

The invention relates to microbiology, in particular to the production of culture media, and can be used in medicine and veterinary medicine in the diagnosis of tuberculosis.

The Levenshtein-Jensen medium is known, including saline (potassium phosphate - 2.4 g, magnesium sulfate - 0.34 g, magnesium citrate - 0.6 g, asparagine - 3.6 g, glycerin - 12 ml, distilled water - up to 600 ml), egg mass - 1000 ml (20-25 eggs), an aqueous 2% solution of malachite greens - 20 ml, potato starch - 30 g ("Manual for the diagnosis of tuberculosis". - M., 2002. - S. 47 is a prototype).

However, using this medium, it is not always possible to isolate bovine tuberculosis mycobacteria, and in the case of positive results, the growth of cultures is visually detected on days 20-60, which lengthens the time for diagnosis of tuberculosis. In addition, due to the evolutionary variability of the causative agent of tuberculosis, the uncontrolled use of drugs and biological products, the information content of bacteriological studies in tuberculosis has decreased.

It is necessary to develop a nutrient medium that provides microscopically visible growth of cultures of mycobacteria at an earlier date for an accelerated diagnosis of tuberculosis and timely measures to eliminate the infection.

The specified technical result is achieved in that the nutrient medium for the cultivation of mycobacteria in the bacteriological diagnosis of tuberculosis (including saline, egg mass, an aqueous solution of malachite greens and potato starch) additionally contains an aqueous extract from the powder of the tail of the deer with the following contents of the components of the medium:

- saline solution:

potassium phosphate - 2 g,

magnesium sulfate - 0.28 g,

magnesium citrate - 0.5 g,

asparagine - 3.0 g

glycerin - 10 ml

distilled water - 500 ml;

- water extract from the tails of maral - 100 ml,

- egg mass - 1000 ml,

- aqueous 2% solution of malachite greens - 20 ml,

- potato starch - 30 g.

In this case, water extraction from the powder of the tailings is carried out by diluting the powder: water 1:10 for 2-3 hours at a temperature of 95.5 ° C and atmospheric pressure.

The basis for studying the possibility of using the maral tail extract as an effective stimulator of the growth of bovine tuberculosis mycobacteria was the data on the comparative biochemical composition of various types of bio products of antler reindeer husbandry, presented in table 1.

Table 1 Biochemical composition Antlers of the maral Sika deer antlers Maral blood Sika deer blood Maral meat Tails Fruit Penis one 2 3 four 5 6 7 8 9 Amino acids g / kg Isoleucine 0.40 1.05 1.28 0.26 2.69 3.25 1.48 2.84 Threonine 0.42 2.12 2.59 0.14 1.82 2.24 1,69 1.88 Series 0.63 0.69 1.25 0,03 1.27 1.39 1,0 1.29 Glycine 0.79 0.85 1.34 0.21 1.36 1.47 1.09 1.37 Alanya 0.81 0.98 1,67 0.46 1.77 1.97 1.36 1.79 Valine 0.58 0.77 1.46 0.83 1,53 1.70 1.13 1,54 Methionine 0.08 0.89 1.10 0.17 0.76 0.95 0.70 0.78 Leucine 0.26 1.38 3.14 0.79 5.38 6.39 2.71 5.43 Glutamine 1.31 2.15 4.46 1.44 4.96 5.52 3.74 4.83

Proline - 0.26 1.93 - 2,32 2.95 1.01 2,33 Phenylalanine 0.28 0.49 1,11 0.24 1.23 1.40 0.88 1.22 Lysine 0.87 3.23 3.87 0.60 2.80 3.39 2.63 2.88 Arginine 0.86 0.87 1.40 0.16 2.20 2.42 1.25 2,32 Tryptophan - 0.44 - - 0.35 0.48 0.32 0.37 Oxyproline - 0.04 - - 0,03 0,03 0.02 0,03 Amount of Amino Acids 7.03 16,20 26.60 5.33 31.80 37.30 22,20 32.30 Vitamins A, million I.E. - - - 34.3 36.6 35.7 30.9 31.3 E, mg / kg 0.58 0.60 0.11 0.45 12.21 11.83 10.28 10.46 B ₁ mg / kg 0.58 0.60 0.10 0.10 1.22 1.18 1,03 1.05 B ₂ mg / kg 1.75 1.80 0.33 3.06 3.66 3,55 3.84 3.14 B ₃ mg / kg 2,57 2.82 0.47 3.85 5.68 5.65 4.90 4.84 ₅ mg / kg 8.78 9.63 1,62 13.15 64.50 64.0 55.70 55.00 B ₆ mg / kg 1.14 - - 2.04 2.44 2,36 2.25 2.1 B ₁₂ mg / kg 5.83 6.00 1.12 5.04 12,20 11.80 10.30 10.50 Mineral composition Ash% 41,4 38.14 - - 3.69 2,32 15.29 2.73 Calcium% 12,4 12.5 10.50 10.80 0.05 0.11 1,60 0.06 Phosphorus,% 7.34 4.01 5.74 7.99 0.60 0.36 1.34 0.41 Potassium g / kg 4.41 3.70 2.25 6.50 12.0 5,0 16,0 6.0 Sodium, g / kg 8.42 7.50 1.33 7.29 2,50 3.30 31.7 5,0 Magnesium g / kg 2.0 1.24 0,07 0.15 0.96 0.65 1.17 0.69 Iron, mg / kg 235.6 675.0 1000,0 1812.0 276.0 130.0 270,0 110.0 Manganese, mg / kg 10.9 8.0 0.17 0.33 1.70 3.30 8.30 1.70 Copper mg / kg 9.68 6.90 2,50 3,7 1,0 4.60 25.0 3,7 Zinc mg / kg 60.9 71.20 6.90 10.0 30,0 25.0 27.5 27.5

As can be seen from table 1, the indicator of amino acids and vitamins is most significant in the tails of deer, where such amino acids as glycerin, alanine, leucine, and glutamine (an available source of nitrogen) are in priority quantities for the growth and development of mycobacteria. In addition, in the manufacture of aqueous extracts from the tailings, they are enriched in fats and phospholipids, which in total provide the energy potential for the life support of microorganisms.

In addition, it should be noted that mycobacteria are very resistant to chemicals and environmental factors due to the presence of fatty substances in the microbial cell. Mycobacterium tuberculosis, unlike other microorganisms, is characterized by a high lipid content, reaching 10-14% of the dry matter of mycobacteria, while phospholipids comprise about 20% of all lipids of mycobacteria.

Research on the lipid content in tail tissue is 3.8%, while in antlers this indicator was at the level of 0.73%, i.e. 5.2 times less.

At the same time, phospholipids account for 39.6% of the total lipids in the tail glands. In the growing cells of mycobacteria, both synthesis and cleavage of phospholipids, which are essential structural elements of the cell walls of mycobacteria, occur. It is known that when a cell uses the entire substrate of a nutrient medium, it begins to consume internal reserves (glycerides, waxes, etc.). Thus, with an increase in the lipid component of the nutrient medium (a modified component of the Levenshtein-Jensen medium), a higher content of amino acids and vitamins, the declared nutrient medium should provide a higher energy potential for the life support of mycobacteria. The mineral composition of tailings extracts, as shown by further studies, turned out to be optimal in terms of the number of necessary macro- and microelements to achieve (the whole set of biochemical components in the extract) accelerated cultivation of mycobacteria. The specific gravity of these extractable substances is 24-25% per 100 g of dry weight.

Based on preliminary studies, an experiment was conducted to identify the optimal parameters of the extraction process of maral tail powder from powder.

A positive effect of the degree of dilution and extraction time on the yield of dry matter in the extraction process at atmospheric pressure and a gentle temperature of 99.5 ° C was revealed. At higher temperatures and pressures, a decrease in the vitamin content was observed. The results of the influence of the parameters of the process of extraction of maral tail powder from the powder on the percentage of dry matter yield are shown in table 2.

table 2 Indicator The time of the process at a ratio of powder (from tailings): water, hour one 2 3 1: 5 1:10 1:15 1: 5 1:10 1:15 1: 5 1:10 1:15 Dry matter 16.50 22,20 21.10 17.40 23.40 22.10 19.80 24,250 23.30

As can be seen from table 2, with 2- and 3-hour extraction, a high yield of dry matter was observed at a dilution of 1:10 (23.4-24.25%) and 1:15 (22.1-23.3%) - optimal 2–3-hour extraction at 1:10 dilution, at atmospheric pressure, and a temperature of 99.5 ° C should be considered the mode of water extraction of powder from tails. The yield of dry matter is at the level of 23.4-24.25%, i.e. at the extremely close level to the maximum possible (24-25%). In general, it should be noted that the biochemical composition of the extract from the tails of maral meets the requirements for raw materials for the production of a colloidal base of a dense nutrient medium in the presence of substances necessary to accelerate the cultivation of mycobacteria.

A nutrient medium with an aqueous extract from the tails of maral was tested on a sample of biomaterial taken from a maral that was reactive to tuberculin and killed for diagnostic purposes, and its autopsy revealed in the internal organs and lymph nodes changes characteristic of tuberculosis in the form of purulent-necrotic abscesses surrounded by connective tissue -woven capsule.

Table 3 shows comparative tests of nutrient media (growth pattern of mycobacteria) according to the prototype (Levenshtein-Jensen medium) and nutrient mixture according to the claimed invention (medium based on the extract from the tails of maral).

The research results indicate that, in terms of primary growth, the nutrient medium with the extract from the tails of maral exceeds the prototype by 4 times, since the growth of colonies of bovine tuberculosis mycobacteria in the subculture was detected on this medium already on day 5, while in the prototype - 20 days.

The average growth rates for large colonies in both compared media are 1. However, the average colonies grew 3.18 times and small 14.1 times more on a nutrient medium with an extract from maral tails.

Size parameters in absolute digital indicators of the studied colonies of mycobacteria differ insignificantly both in the prototype and in the nutrient medium based on the maral tail extract, but, at the same time, the seeding rate is 25% higher on the medium using the maral tail extract. The growth of banal microflora was not detected in both compared media.

Example 1. A nutrient medium for the cultivation of mycobacteria in the bacteriological diagnosis of tuberculosis is prepared as follows.

1) First, according to the “Manual for the diagnosis of tuberculosis”. - M., 2002. - P.47, prepared a saline solution of Levenshtein-Jensen medium consisting of:

- potassium phosphate - 2.4 g;

- magnesium sulfate - 0.34 g;

- magnesium citrate - 0.6 g;

- L-asparagine - 3.6 g;

- glycerol - 12 ml;

- distilled water up to 600 ml.

2) Next, an extract was prepared from the powder of maral tail. Powder from the tails (particle size 100-400 μm) was placed in the working capacity of the extractor and poured with water in the ratio of powder: water 1:10. The extraction was carried out at a temperature close to 100 ° C (99.5 ° C) under atmospheric pressure for 3 hours. The contents of the container were filtered through a gauze filter, followed by sterilization (20 min) at the same temperature.

3) 500 ml of saline solution was mixed with 100 ml of extract from the tails of maral and all remaining components of the Levenshtein-Jensen medium were added:

- egg mass of 1000 ml (20-25 eggs);

- aqueous 2% solution of malachite greens - 20 ml;

- potato starch - 30 g.

After the ingredients were completely dissolved, pH 7.6 was set (initially at pH 5.7 for extraction), after which the mixture was filtered, poured into test tubes (4-5 ml each), placed in an inclined position in a medium coagulator, sterilized at 85 ° C for 30 minutes.

From the selected biomaterial (affected organs and lymph nodes from the killed for diagnostic purposes, reacting to tuberculin deer), processed according to the method of Gon-Levenshtein-Sumyoshi, inoculation was performed on medium according to the prototype and nutrient medium according to the claimed method (medium with extract from the tail of maral) . The results are shown in table 4.

Table 4 Levenshtein-Jensen medium (prototype) Primary growth in a biomaterial culture, days Colony growth rate, qty The size of the colonies,% Number of test tubes Crop growth The growth of banal microflora to from m to from m test tubes % test tubes % 41-55 - - twenty - - one hundred 60 32 53.3 - - Culture medium based on the extract of maral tail Primary growth in a biomaterial culture, days Growth rate, colonies, pcs The size of the colonies,% Number of test tubes Crop growth The growth of banal microflora to from m to from m test tubes % test tubes % 8-11 one 2.0 19,4 0.001 2.7 97.2 60 57 95 - -

As can be seen from table 4, the primary growth of cultures of mycobacteria (according to the results of cultural-morphological and biological studies attributed to bovine tuberculosis mycobacteria) according to the prototype is 41-55 days, and on a nutrient medium with an extract from the tails of maral - 8-11 days, which made it possible to diagnose tuberculosis 33 days earlier and start preventive measures to eliminate it in maral breeding.

The growth medium nutrient medium with the extract from the tails of maral exceeded the prototype 9.7 times in growth of small colonies, in 2 and 1 in medium and large.

Of the 60 test tubes, the growth of cultures on a nutrient medium with extract from the tails of maral was detected in 95% of the tubes, at the same time, according to prototype 0, in 53.3%, which is 41.7% less.

Example 2. Due to the fact that the withdrawal of 100 ml of the saline solution is replaced (as part of the saline solution of Levenshtein-Jensen medium) during the preparation of the claimed nutrient medium with an extract from the tail powder, some losses of the saline solution components should be noted (especially in the manufacture of environment). Therefore, it is possible to prepare immediately not 600, but 500 ml of the saline solution used in the claimed medium while maintaining the previous concentration of the components. In this case, the content of the components of the saline solution is as follows:

- potassium phosphate - 2.0 g;

- magnesium sulfate - 0.28 g;

- magnesium citrate - 0.5 g;

- L-asparagine - 3.0 g;

- glycerin - 10 ml;

- distilled water up to 500 ml.

The remaining components of the medium in the same composition and quantity as in example 1.

All subsequent text is also on the 1st example.

Thus, the use of the claimed nutrient medium for the cultivation of mycobacteria in the bacteriological diagnosis of tuberculosis can reduce the isolation time of mycobacterium tuberculosis, which provides an accelerated diagnosis of animal tuberculosis, and therefore timely measures to eliminate the infection.

Claims (1)

Culture medium for the cultivation of Mycobacterium tuberculosis containing potassium phosphate, magnesium sulfate, magnesium citrate, L-asparagine, glycerin, potato starch, distilled water, egg mass, 2% solution of malachite green, characterized in that in addition to the mixture of these components, the medium contains an aqueous extract of powder from the tails of maral, prepared by extracting the powder with water in a ratio of 1:10, respectively, at a temperature of 95.5-99.5 ° C and atmospheric pressure for 2-3 hours, the following contains and components:
potassium phosphate 2.0 g magnesium sulfate 0.28 g magnesium citrate 0.5 g L-asparagine 3.0 g glycerol 10 ml potato starch 30 g distilled water up to 500 ml water extract of maral tail powder 100 ml aqueous 2% solution of malachite green 20 ml egg mass 1000 ml